Discovery of a Highly Potent, Selective, and Orally Bioavailable Macrocyclic Inhibitor of Blood Coagulation Factor VIIa-Tissue Factor Complex

Article abstract of DOI:10.1021/acs.jmedchem.6b00469

Inhibitors of the tissue factor (TF)/factor VIIa complex (TF-FVIIa) are promising novel anticoagulants which show excellent efficacy and minimal bleeding in preclinical models. Starting with an aminoisoquinoline P1-based macrocyclic inhibitor, optimizatio

Full text of DOI:10.1021/acs.jmedchem.6b00469

Article
pubs.acs.org/jmc
Discovery of a Highly Potent, Selective, and Orally Bioavailable
Macrocyclic Inhibitor of Blood Coagulation Factor VIIa−Tissue Factor
Complex
Xiaojun Zhang,*,† Peter W. Glunz,† James A. Johnson,† Wen Jiang,† Swanee Jacutin-Porte,†
Vladimir Ladziata,† Yan Zou,†,§ Monique S. Phillips,† Nicholas R. Wurtz,† Brandon Parkhurst,†
Alan R. Rendina,‡ Timothy M. Harper,‡ Daniel L. Cheney,† Joseph M. Luettgen,‡ Pancras C. Wong,‡
Dietmar Seiffert,‡ Ruth R. Wexler,† and E. Scott Priestley†
†Bristol-Myers Squibb R&D, 350 Carter Road, Hopewell, New Jersey 08540, United States
‡Bristol-Myers Squibb R&D, 311 Pennington-Rocky Hill Road, Pennington, New Jersey 08534-2130, United States
S
* Supporting Information
ABSTRACT: Inhibitors of the tissue factor (TF)/factor VIIa complex (TF-FVIIa) are
promising novel anticoagulants which show excellent efficacy and minimal bleeding in
preclinical models. Starting with an aminoisoquinoline P1-based macrocyclic inhibitor,
optimization of the P′ groups led to a series of highly potent and selective TF-FVIIa inhibitors
which displayed poor permeability. Fluorination of the aminoisoquinoline reduced the basicity
of the P1 group and significantly improved permeability. The resulting lead compound was
highly potent, selective, and achieved good pharmacokinetics in dogs with oral dosing.
Moreover, it demonstrated robust antithrombotic activity in a rabbit model of arterial
thrombosis.
P1 group (pKa ∼ 12).16 The basic group engages in a strong
INTRODUCTION
■
interaction with Asp189 in the active site of FVIIa, resulting in
Pathological blood clotting initiated by tissue factor (TF) leads
inhibitors that are potent, but poorly permeable/orally
to thromboembolic cardiovascular diseases such as ischemic
stroke, myocardial infarction, and deep venous thrombosis.1
bioavailable. Alternatively, several groups have attempted to
replace the benzamidine moiety with less basic groups,
Atherosclerotic plaque rupture or vascular injury exposes TF to
circulating coagulation factor VIIa (FVIIa), a serine protease.
Binding of TF to FVIIa and formation of the TF-FVIIa complex
although with limited success.17−23
Our group reported a series of aminoisoquinoline-based
phenylpyrrolidine phenylglycineamides as potent and selective
leads to a large increase in its catalytic activity and subsequently
TF-FVIIa inhibitors displaying promising oral bioavailabilty.24
initiates the extrinsic coagulation pathway by activating factor
On the basis of a crystal structure of a phenylpyrrolidine lead
IX to IXa and factor X to Xa. Factor Xa then activates
prothrombin to thrombin.2 Thrombin plays an essential role in
bound in the active site of FVIIa and subsequent molecular
modeling studies, Priestley et al. recently reported a novel series
the pathogenesis of thrombosis by cleaving fibrinogen to fibrin
of macrocyclic TF-FVIIa inhibitors with improved potency
and activating platelets through proteolytic activation of
relative to the acyclic analogues.25 Further potency optimiza-
tion and structure guided atropisomer control by incorporation
of P2 methyls provided a macrocyclic compound 1 with TF-
FVIIa Ki (37 °C) of 20 nM and measurable PAMPA
permeability of 13 nm/s (Figure 1).26 Discovery of the
conformationally constrained macrocyclic scaffold represented
a significant advance in the search for potent TF-FVIIa
inhibitors and opened new possibilities for orally bioavailable
TF-FVIIa inhibitor development. Compound 1, although
equally potent against human tissue kallikrein (HK-1, Ki = 21
nM), served as an attractive starting point for further
improvement. Herein we report optimization of the P′ groups
and fluorination of the aminoisoquinoline P1 group in 1 that
thrombin receptors, enabling clot formation.3,4 It has been
shown that selective inhibition of the TF-FVIIa complex
provides antithrombotic efficacy with a low risk of bleeding in
preclinical models.5−13 In addition, recombinant nematode
anticoagulant protein c2, a biological agent that inhibits TF-
VIIa complex, demonstrated the antithrombotic efficacy and
safety in patients with acute coronary syndrome.14 Accordingly,
direct-acting, orally bioavailable TF-FVIIa inhibitors could be
effective and safe in the treatment and prevention of venous
and arterial thrombosis. Despite strong target validation and
substantial efforts to discover orally active TF-FVIIa inhib-
itors,15 there are no reports of small molecule TF-FVIIa
inhibitors advancing to antithrombotic clinical trials. The most
significant unsolved issue for small molecule drugs targeting
TF-FVIIa is oral bioavailability; the vast majority of known TF-
FVIIa inhibitors contain benzamidine or a similar highly basic
Received: March 30, 2016
© XXXX American Chemical Society
A
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Figure 1. Optimization of P′ and fluorination of P1 in 1 led to the discovery of lead compound 13.
a
Scheme 1. Synthesis of Macrocyclic Inhibitors 1 and 8−10
a
Reagents and conditions: (a) DMF/CH3CN (1:2), 80 °C; (b) BOP, DIEA, DMF/CH3CN (1:2), 51−77% for 2 steps; (c) phosgene, CH3CN/
CH2Cl2 (1:1), then TEA; (d) chiral HPLC, 37−49% for 2 steps; (e) TFA/CH2Cl2 or 4.0 N HCl in dioxane/EtOAc, 36−60%; (f) LiOH, THF, 40
°C, 51%.
a
Scheme 2. Synthesis of Macrocyclic Inhibitors 2 and 7
a
Reagents and conditions: (a) BOP, DIEA, DMF/CH3CN (1:2), 54−59%; (b) Pd/C, H2 balloon, MeOH, 82−89%; (c) phosgene, CH3CN/CH2Cl2
(1:1), then TEA; (d) chiral HPLC, 12−32% for 2 steps; (e) TFA/CH2Cl2, 44−90%.
led to the discovery of the lead compound 13, a highly potent,
selective macrocyclic inhibitor of TF-FVIIa with good
pharmacokinetics in dogs from oral dosing and robust
antithrombotic activity in a rabbit model of arterial thrombosis.
ypropan-2-yl)-3-methylphenyl)boronic acid 16,29 and glyox-
ylate 17 gave rise to the phenylglycine derivative 18. Coupling
of intermediate 18 with substituted N-methylbenzylamines
19a−c afforded the corresponding phenylglycinamides 20a−c.
The Petasis reaction and amide coupling could be conveniently
carried out in a two-step, one-pot procedure without isolation
of the acid 18. Macrocylization to form the upper carbamate
bond was accomplished by the treatment of compounds 20a−c,
first with phosgene to generate a transient chloroformate,
CHEMISTRY
■
The syntheses of the macrocyclic inhibitors 1 and 8−10 are
outlined in Scheme 1. A three-component Petasis reaction27 of
di-Boc protected 6-aminoisoqunoline 15,28 (R)-(4-(1-hydrox-
B
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a
Scheme 3. Synthesis of Macrocyclic Inhibitors 3−6
a
Reagents and conditions: (a) Pd(OAc)2, dppp, CO (25 psi), TEA, DMSO/MeOH (2:1), 80 °C, 83%; (b) LiOH, THF/H2O (2:1), 57%; (c) EDC/
HOBt, NH4Cl, DIEA, 97% for 4; HATU, N-methylmorpholine, DMF, 29−39% for 5−6; (d) TFA, 80−90%.
a
Scheme 4. Synthesis of Macrocyclic Inhibitors 11−13
a
Reagents and conditions: (a) DMF/CH3CN (1:2), 80 °C; (b) BOP, TEA, DMF/CH3CN (1:2), 38−54% for two steps; (c) phosgene, CH3CN/
CH2Cl2 (1:1), then TEA; (d) chiral HPLC, 34−43% for 2 steps; (e) TFA/CH2Cl2 or 4.0 N HCl in dioxane/EtOAc, 17−90%.
followed by a slow addition of the chloroformate to
triethylamine in dichloromethane to finish the lactamization.26
At this stage, the two diastereoisomers were separated by chiral
HPLC to yield the desired enantiomer. Deprotection of the di-
Boc group with acid and a final HPLC purification afforded the
macrocyclic inhibitors 1, 8, and 10. Macrocyclic inhibitor 9 was
obtained by a monohydrolysis of the diethylphosphonate 8
with LiOH. The Petasis reaction/amide coupling provided an
efficient and modular synthetic route to macrocyclic TF-VIIa
inhibitors.
Syntheses of macrocyclic inhibitors 2 and 7 are shown in
Scheme 2. Nitro substituted N-methylbenzylamines 21a,b were
coupled with intermediate 18. After amide coupling, the nitro
group was readily reduced to give anilines 20d,e. Macro-
cyclization, chiral separation, and deprotection of the di-Boc
group as exemplified in Scheme 1 yielded macrocyclic
inhibitors 2 and 7.
Syntheses of macrocyclic inhibitors 3−6 were achieved by
late stage functionalization of an advanced intermediate, as
shown in Scheme 3. The macrocyclic bromide 22 was subjected
to a Pd-catalyzed carbonylation to give rise to the methyl ester
23. Deprotection of the di-Boc group in 23 yielded inhibitor 3.
Methyl ester 23 was hydrolyzed to the corresponding acid 24.
Amide coupling of the acid 24 with amines, followed by
deprotection of the di-Boc group, afforded macrocyclic
inhibitors 4-6.
Syntheses of macrocyclic inhibitors 11−13 are shown in
Scheme 4. Petasis reaction of a fluoro-substituted amino-
isoquinoline 25a−c, boronic acid 16, and glyoxylate 17 gave
rise to the phenylglycine derivatives 26a−c. Coupling of 26a−c
with 4-(cyclopropylsulfonyl)-3-((methylamino)methyl)aniline
19c afforded phenylglycinamides 27a−c. It is advantageous to
carry out the petasis reaction/amide coupling in a two-step,
one-pot procedure without isolation of the acid 26. Macro-
cylization, chiral separation, and di-Boc deprotection, as
described for the synthesis of compounds 1−10, yielded
macrocyclic inhibitors 11−13.
Syntheses of fluoro-substituted aminoisoquinolines 25a−c
are summarized in Scheme 5. 8- or 7-Fluoro substituted 6-
(dibenzylamino)isoquinolin-1(2H)-one 2828 was treated with
POCl3 to generate the corresponding chloride, which was
reacted with ammonia in ethylene glycol to yield 8- or 7-fluoro
N,N-dibenzylisoquinoline-1,6-diamine 29. The displacement of
the chloride by ammonia had to be carried out at high
temperature in an autoclave to obtain moderate yields of the
aminoisoquinolines 29a,b. Protection of the free amines 29a
and 29b with Boc anhydride, followed by Pd-catalyzed
hydrogenation with Pearlman’s catalyst in ethanol, gave rise
C
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to the corresponding 8- and 7-fluoroaminoisoquinolines 25a
and 25b in good yield.
Scheme 5. Synthesis of Fluoro-Substituted
Aminoisoquinolines 25a−c
a
A different strategy was devised to prepare the 4-
fluoroaminoisoquinoline 25c. Compound 30, prepared in
high yield as previously described,28 reacted with Selectfluor
in methanol to generate methanol-trapped fluorine adduct 31
in a nearly quantitative yield. Treatment of hemiaminal 31 with
HCl in acetonitrile resulted in elimination of methanol and
aromatization to afford 4-fluoro-6-nitroisoquinolin-1(2H)-one
32 in 98% yield. Compound 32 was converted to the
corresponding chloride 33 with POCl3. Displacement of the
chloride 33 by ammonia to give amine 34 proved to be
problematic because of partial displacement of fluoride.
Consequently, Pd-catalyzed amination of the chloride 33 with
benzophenone imine, followed by treatment with acid, yielded
the 4-fluoro-6-nitroisoquinolin-1-amine 34 in good yield. Boc
protection of 34 and reduction of the nitro group afforded 4-
fluoroaminoisoquinoline 25c. The optimized sequence enabled
a high yielding and scalable synthesis of the key intermediate
25c.
a
Reagents and conditions: (a) POCl3, 100 °C, >95%; (b) NH3,
HO(CH2)2OH, 130 °C, 50−64%; (c) (Boc)2O, DMAP (cat.),
CH3CN, 66−77%; (d) Pd(OH)2, H2 (55 psi), EtOH, 90−96%; (e)
Selectfluor, MeOH/CH3CN (1:1), 82 °C, 99%; (f) 4.0 HCl, dioxane/
CH3CN (1:1), 65 °C, 98%; (g) Pd(OAc)2, benzophenone imine,
BINAP, Cs2CO3, toluene, 90 °C, then HCl, THF/H2O, 79%; (h) Pd/
C, H2 (balloon), HCl (cat.), MeOH, 99%.
RESULTS AND DISCUSSION
■
Our strategy to improve the potency of macrocyclic inhibitor 1
was to optimize the molecular interactions of the inhibitors
with the S′ site of FVIIa, in an analogy to that of the
phenylpyrrolidine phenylglycineamide series,24 by substitution
at the ortho position (to the benzyl amide) of the P′ phenyl
Table 1. Summary of P′ SAR of the Macrocyclic TF-FVIIa Inhibitors
a
b
TF-FVIIa assays were performed at 37 °C (n ≥ 2). Kis for the indicated enzymes were determined at 25 °C, except HK-1, which was run at 37 °C
c
d
(n = 2). See ref 11 for a detailed description of the FVII deficient prothrombin time assay. Not determined
D
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ring (Table 1). The in vitro activity was expressed as the
inhibition constant Ki against human TF-FVIIa at 37 °C with
physiologically relevant factor X (FX) as the substrate.11 The
selectivity of the compounds was first evaluated against a panel
of serine proteases including coagulation factor Xa (FXa),
factor XIa (FXIa), thrombin, trypsin, and human tissue
kallikrein (HK-1). For compounds of interest, selectivity was
evaluated with a broader range of serine proteases.11 The
anticlotting activity of the compounds was evaluated with a
FVII deficient prothrombin time (FVII def PT) assay in human
plasma, expressed as the concentration required to prolong
clotting time by 2-fold (EC2x).11 The permeability of the
macrocyclic compounds was evaluated in a parallel artificial
membrane permeability assay (PAMPA).30
Compound 1 showed a promising overall profile. It exhibited
good potency and anticlotting activity (Ki = 20 nM, EC2x = 5.3
μM). It displayed excellent selectivity against FXa, FXIa,
thrombin, and trypsin. However, it retained an equal potency
against HK-1 (Ki = 21 nM) and displayed low but measurable
PAMPA permeability (Pc = 13 nm/s). As shown in Table 1, the
trifluoromethoxy group in compound 2 improved the binding
affinity for TF-FVIIa by 4-fold and maintained good overall
selectivity except for HK-1. The anticlotting activity of
compound 2 (EC2x = 10 μM) was 2-fold less potent than
that of compound 1 despite its improved binding affinity. This
is most likely due to its decreased free fraction in human plasma
as a result of increased lipophilicity compared to compound 1.
The trifluoromethoxy moiety significantly improved the
PAMPA permeability (Pc = 230 nm/s) in compound 2.
Although methyl ester 3 and primary amide 4 only slightly
improved the TF-FVIIa binding affinity, the selectivity against
HK-1 of the two compounds was significantly improved while
the excellent selectivity profile against other coagulation serine
proteases was maintained. N,N-Dimethyl and N,N-diethyl
amide 5 and 6 further improved the selectivity against HK-1
by concomitantly improving the binding affinity to TF-FVIIa
and reducing the binding affinity to HK-1. Compound 6 proved
to be a highly potent (Ki = 0.57 nM) and selective (>1000-fold)
TF-VIIa inhibitor with good anticlotting activity (EC2x = 2.2
μM). Unfortunately, the PAMPA permeability of compound 6
(Pc = 33 nm/s) was only modest compared to 1.
Small heterocycles such as pyrazole 7 improved the TF-FVIIa
activity as well as the anticlotting activity with moderate
permeability compared to compound 1. Diethyl phosphate 8
and ethyl phosphonic acid 9 also significantly improved the TF-
VIIa binding affinity and anticlotting activity. Compound 9
displayed an exceptional selectivity profile including against
HK-1, suggesting a polar group in the P′ site is beneficial for
achieving good HK-1 selectivity. Not surprisingly, the high
polarity of compound 9 significantly limited its permeability
(Pc = 3 nm/s). The most profound improvement in potency
originated from the substitution of cyclopropyl sulfone in the P′
site, affording compound 10 with an excellent TF-FVIIa
Figure 2. Model of 13 bound in FVIIa. Graphics were generated with
the program PyMOL.35
structures of analogous molecules.34 In this model, the fluoro-
aminoisoquinoline forms a salt bridge with Asp189 side chain
and a hydrogen bond with a water molecule above Tyr228 side
chain. In the S1′ pocket, the amide carbonyl forms a pair of
hydrogen bonds to the catalytic Ser195 and H57 side chains.
The phenyl sulfone directs the cyclopropyl ring toward the
Cys42−Cys58 disulfide bridge.
Compound 10 showed good metabolic stability in human
and dog liver microsomes with t1/2 > 100 min but limited
metabolic stability in rat liver microsomes with t1/2 = 12 min
(Table 2). Given its excellent potency and selectivity, good
anticlotting activity, and moderate PAMPA permeability,
compound 10 was progressed to dog pharmacokinetic studies.
To our disappointment, it demonstrated very low oral exposure
with only 1% absolute oral bioavailability. We believe its limited
oral bioavailability is most likely attributed to its insufficient
permeability. Despite modest potency, compound 2 possessed
significantly improved PAMPA permeability and good meta-
bolic stability in human and dog liver microsomes. Accordingly,
it achieved much higher oral exposure than compound 10 with
a 22% oral bioavailability in dogs. These results strongly
suggested that good oral exposure of compound 10 could be
achieved by improvement of its PAMPA permeability.
It is well established that the strong electron withdrawing
nature of fluorine can exert significant effects on the basicity of
neighboring functional groups.32 We decided to investigate
fluoro substitution on the aminoisoquinoline P1 in macrocyclic
inhibitor 10 as a strategy to reduce the basicity and thus
improve permeability (Table 3). The unsubstituted amino-
isoquinoline 10 has an experimentally determined pKa of 8.7
and calculated pKa of 8.8.33 Compound 11, with fluoro
substitution at the 8-position, has a measured pKa of 8.0 relative
to 8.7 for compound 10. As expected, the reduced basicity
translated into improved PAMPA permeability (from 50 nm/s
in compound 10 to 200 nm/s in compound 11). The reduction
of basicity in 11 by fluorination resulted in 6-fold loss of
binding affinity to TF-FVIIa and about 3-fold loss of
anticlotting activity compared to 10. On the other hand,
compound 11 improved binding affinity to HK-1 by nearly 3-
fold, resulting in a less selective profile. Compound 12, with
fluoro substitution at the 7-position, has a calculated pKa of 8.5,
marginally reduced relative to 8.8 in compound 10.33 It resulted
in a compound of comparable inhibitory potency relative to 10
but still with improved PAMPA permeability (Pc = 180 nm/s).
binding affinity and anticlotting activity (Ki = 0.16 nM, EC2x
=
1.1 μM) while still maintaining an excellent selectivity profile
(>1600-fold) and moderate permeability (Pc = 50 nm/s). This
interaction, which yielded more than 120-fold potency
improvement relative to compound 1, was observed earlier in
the acylsulfonamide and phenylpyrrolidine phenylglycinamide
series.24,31 It is believed that the cyclopropylsulfone engages in
hydrophobic interaction with Cys42−Cys58 disulfide bridge in
the active site of TF-FVIIa complex. Figure 2 depicts a binding
model of 13 bound in FVIIa based on crystallographic
E
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Table 2. Metabolic Stability, Permeability, and Oral Bioavailability of Compounds 10 and 2
microsomal stability t1/2 (min)
a
compd
human
dog
rat
PAMPA Pc (nm/s)
oral bioavailability in dogs (F%)
10
2
>120
130
110
140
12
37
50
1
230
22
a
Dogs (n = 3) were dosed with 0.2 mg/kg iv and 1.0 mg/kg po.
with t1/2 = 8 h and F% = 40. Thus, the 4-fluoroaminoisoquino-
line had the most profound impact on pKa, permeability, and
oral bioavailability in this series of macrocyclic TF-FVIIa
inhibitors. Because of its poor metabolic stability in rat and
mouse liver microsomes (t1/2 = 1.9 and 3.1 min, respectively),
compound 13 showed poor oral exposure in both species with
absolute oral bioavailability <5%.
Table 3. Effects of Fluorine Substitution on P1
Because of its superior pharmacokinetic properties in dogs,
compound 13 was selected for further in vitro and in vivo
characterization. In addition to its excellent TF-FVIIa inhibitory
activity, it displayed excellent selectivity toward other proteases
important to hemostasis and thrombolysis (Table 5). It showed
F
FVIIa
Ki
HK-1
Ki
FVII def
PT EC2x
(μM)
PAMPA
Pc
position
on P1
a
compd
pKa
(nM)
(nM)
(nm/s)
b
10
11
12
13
NA
8.7 (8.8)
8.0 (8.2)
(8.5)
0.16
1.0
270
100
790
650
1.1
2.8
4.2
6.1
50
200
180
900
8-F
7-F
4-F
Table 5. Selectivity Profile of Macrocyclic Inhibitor 13
0.23
0.43
a
human enzyme
TF-FVIIa
Ki (nM)
selectivity (fold)
7.1 (7.5)
b
a
0.43
1600
4000
3400
7500
11000
3000
520
NA
Data in parentheses refers to QM calculated pKa. See ref 33 for
details. Not applicable.
c
b
FIXa
>3700
>9300
>7900
>17000
>26000
>7000
>1200
440
FXa
FXIa
Fluoro substitution at the 4-position had the most profound
impact on pKa and permeability. Compound 13 achieved nearly
two log units reduction of the measured pKa (from 8.7 to 7.1)
and 18-fold improvement of PAMPA permeability (from 50 to
900 nm/s) relative to 10 while maintaining excellent TF-FVIIa
inhibitory activity and good anticlotting activity (Ki = 0.43 nM,
EC2x = 6.1 μM). The data suggests that 4-fluoroaminoisoquino-
line is still capable of strong binding in the S1 specificity pocket
of TF-FVIIa via ionic interaction with Asp189 despite its
significantly reduced basicity.
d
FXIIa
thrombin
trypsin
plasma kallikrein
e
aPC
190
HK-1
650
>1500
>39000
>3500
>21000
>35000
chymotrypsin
plasmin
17000
1500
9100
15000
f
tPA
urokinase
The fluorinated macrocyclic inhibitors 11−13 showed good
metabolic stability in human liver microsomes but poor
metabolic stability in rat as observed in compound 10 (Table
4). Further pharmacokinetic studies were performed in dogs
because of the good metabolic stability achieved with
compounds 11 and 13.36 Compound 11, with a t1/2 = 39
min in dog liver microsomes and permeability Pc = 200 nm/s,
displayed a moderate clearance and volume of distribution (CL
= 13 mL/min/kg, Vdss = 8 L/kg) with t1/2 = 8 h and F% = 24.
Compound 12, with t1/2 = 7 min in dog liver microsomes,
showed a moderate clearance and low oral exposure, most likely
due to combination of poor metabolic stability and insufficient
permeability. Compound 13, with t1/2 = 26 min in dog liver
microsomes and significantly improved PAMPA permeability
(Pc = 900 nm/s), demonstrated a low clearance and moderate
volume of distribution (CL = 8 mL/min/kg, Vdss = 5 L/kg)
a
TF-FVIIa assays were performed at 37 °C (n > 2). Kis for other
indicated enzymes were determined at 25 °C, except aPC and HK-1,
b
c
d
which were run at 37 °C (n = 2). Not applicable. Factor IXa. Factor
XIIa. Active protein C. Tissue plasminogen activator.
e
f
no significant inhibition in cytochrome P450 enzymes and ion
channels (Na, Ca, and hERG). Free fraction of compound 13
in human and dog plasma was measured to be 2.3% and 1.4%
respectively. Compound 13 exhibited good binding affinity to
rabbit TF-VIIa (Ki = 1.9 nM) with a free fraction of 2.6% in
rabbit plasma. In a rabbit model of electrically induced carotid
arterial thrombosis (ECAT),11 compound 13 inhibited
thrombus formation in a concentration-dependent manner
with an EC50 of 360 nM (Figure 3).
Table 4. Dog Pharmacokinetic Parameters of Macrocyclic Inhibitors 11−13
a
microsomal stability t1/2 (min)
dog PK
compd
human
dog
rat
PAMPA Pc (nm/s)
CL (mL/min/kg)
Vdss (L/kg)
t1/2 (h)
F (%)
11
12
13
120
95
39
7
3.3
10
200
180
900
13
11
8
8
4
5
8
6
8
24
<1
40
45
26
1.9
a
Dogs (n = 3) were dosed with 0.2 mg/kg iv and 1.0 mg/kg po.
F
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chiral HPLC (Whelk-01 25 cm × 21.1 mm, 50% (1:1 MeOH/EtOH)
in heptanes) to give the desired enantiomer (30 mg, 0.083 mmol, 49%
yield). MS (ESI) m/z 710.5 [M + H]+.
Trifluoroacetic acid (2 mL) was added to the above desired
enantiomer (30 mg, 0.083 mmol), and the reaction was stirred at room
temperature for 1 h. The mixture was concentrated and purified by
prep HPLC to yield 1 (19.0 mg, 36% yield) as a white solid. MS (ESI)
1
m/z 510.2 [M + H]+. H NMR (500 MHz, MeOH-d4) δ 8.05 (d, J =
9.35 Hz, 1H), 7.63 (dd, J = 1.79, 7.84 Hz, 1H), 7.43 (d, J = 7.98 Hz,
1H), 7.31 (d, J = 7.15 Hz, 1H), 7.23 (d, J = 1.38 Hz, 1H), 7.21 (dd, J =
2.48, 9.35 Hz, 1H), 7.18 (t, J = 7.84 Hz, 1H), 6.91 (d, J = 6.88 Hz,
2H), 6.84 (d, J = 2.20 Hz, 1H), 6.69 (dd, J = 0.96, 7.84 Hz, 1H), 5.93
(s, 1H), 5.74 (s, 1H), 5.45 (d, J = 16.51 Hz, 1H), 4.61- 4.69 (m, 1H),
3.96−4.03 (m, 1H), 3.91 (d, J = 16.51 Hz, 1H), 3.46−3.55 (m, 1H),
3.29 (s, 3H), 2.34 (s, 3H), 1.31 (d, J = 6.88 Hz, 3H). HPLC purity
100%.
(2R,15R)-2-[(1-Aminoisoquinolin-6-yl)amino]-4,15,17-trimethyl-
7-(trifluoromethoxy)-13-oxa-4,11-diazatricyclo[14.2.2.16,10]-
henicosa-1(18),6,8,10(21),16,19-hexaene-3,12-dione (2). In a proce-
dure similar to that described for 1, 20d (1.21 g, 1.58 mmol) reacted
with phosgene/TEA, diastereoisomers were chirally separated (Chiral
OD 10 μm 4.6 × 250 mm, 40% EtOH/MeOH(50/50) with 0.1%DEA
in heptanes), and the desired enantiomer was deprotected with TFA
to give 2 (240 mg, 41% yield) as a white solid. MS (ESI) m/z 594.7
[M + H]+. 1H NMR (400 MHz, MeOH-d4) δ ppm 9.00 (s, 1H), 7.95
(d, J = 8.79 Hz, 1H), 7.54−7.59 (m, 1H), 7.36 (d, J = 7.70 Hz, 1H),
7.22 (d, J = 7.15 Hz, 1H), 7.09−7.14 (m, 2H), 7.06 (d, J = 7.15 Hz,
1H), 6.82 (d, J = 7.15 Hz, 1H), 6.75 (d, J = 2.20 Hz, 1H), 6.68 (dd, J =
8.79, 2.20 Hz, 1H), 5.94 (d, J = 2.20 Hz, 1H), 5.66 (s, 1H), 5.34 (d, J =
17.59 Hz, 1H), 4.56 (t, J = 10.99 Hz, 1H), 3.80−3.91 (m, 2H), 3.44 (s,
3H), 2.24 (s, 3H), 1.22 (d, J = 7.15 Hz, 3H). 19F NMR (376 MHz,
MeOH-d4) δ ppm −59.11 (s, 3F). HPLC purity 96%.
Methyl (2R,15R)-2-[(1-Aminoisoquinolin-6-yl)amino]-4,15,17-tri-
methyl-3,12-dioxo-13-oxa-4,11-diazatricyclo[14.2.2.16,10]henicosa-
1(18),6,8,10(21),16,19-hexaene-7-carboxylate (3). 23 (15 mg, 0.02
mmol) was dissolved in TFA (1 mL) and stirred for 1 h. The mixture
was concentrated and purified by prep HPLC to yield 3 (11.2 mg, 80%
yield). MS (ESI) m/z 568.2 [M + H]+. 1H NMR (400 MHz, MeOH-
d4) δ ppm 9.32 (s, 1H), 8.04 (d, J = 9.34 Hz, 1H), 7.92 (m, 1H),
7.63−7.71 (m, 1H), 7.47 (m, 1H), 7.31 (m, 1H), 7.20 (dd, J = 9.34,
2.20 Hz, 1H), 6.92 (m, 1H), 6.85 (d, J = 2.20 Hz, 1H), 6.74 (dd, J =
8.79, 2.20 Hz, 1H), 6.21 (br s, 1H), 5.68−5.74 (m, 2H), 4.63 (t, J =
10.99 Hz, 1H), 3.96 (dd, J = 10.99, 4.40 Hz, 1H), 3.79−3.88 (m, 3H),
3.61−3.74 (m, 5H), 2.31 (s, 3H), 1.32 (d, J = 7.15 Hz, 3H). HPLC
purity 95%.
(2R,15R)-2-[(1-Aminoisoquinolin-6-yl)amino]-4,15,17-trimethyl-
3,12-dioxo-13-oxa-4,11- diazatricyclo[14.2.2.16,10]henicosa-1-
(18),6,8,10(21),16,19-hexaene-7-carboxamide (4). 24 (12 mg,
0.016 mmol), EDC (6.1 mg, 0.032 mmol), HOBt (4.9 mg, 0.032
mmol), NH4Cl (1.7 mg, 0.032 mmol), and DIEA (0.011 mL, 0.064
mmol) were added into a vial. To this mixture was added DMF (0.5
mL) and stirred at room temperature under argon overnight. Water
was added, and the reaction mixture was extracted with EtOAc. The
combined organic layers were washed with brine, dried over MgSO4,
and concentrated. The crude product was treated with TFA (1.0 mL)
for 15 min to deprotect the di-Boc group. The mixture was
concentrated and purified by prep HPLC to yield 4 (3.1 mg, 97%
yield) as a white solid. MS (ESI) m/z 553.1 [M + H]+. 1H NMR (400
MHz, MeOH-d4) δ ppm 8.04 (d, J = 9.03 Hz, 1H), 7.66 (d, J = 7.53
Hz, 1H), 7.46 (t, J = 7.28 Hz, 2H), 7.31 (d, J = 7.28 Hz, 1H), 7.20 (dd,
J = 9.16, 2.13 Hz, 1H), 7.16 (s, 1H), 6.91 (d, J = 7.03 Hz, 1H), 6.84 (s,
1H), 6.68−6.78 (m, 1H), 6.17 (br s, 1H), 5.72 (s, 1H), 5.43−5.62 (m,
1H), 4.63 (t, J = 11.04 Hz, 1H), 4.21 (d, J = 17.57 Hz, 1H), 3.98 (dd, J
= 10.54, 4.27 Hz, 1H), 3.58−3.41 (m, 1H), 3.28 (s, 3H), 2.32 (s, 3H),
1.32 (d, J = 7.03 Hz, 3H). HPLC purity 97%.
Figure 3. Antithrombotic effects of compound 13 in the rabbit ECAT
model.
CONCLUSIONS
■
In summary, starting from macrocyclic TF-FVIIa inhibitor 1
and optimizing the interactions with the S′ site, we have
significantly improved potency and selectivity. The cyclopropyl
sulfone 10 showed optimal TF-FVIIa binding affinity and
overall selectivity against a panel of serine proteases. To address
poor permeability and improve oral bioavailability in the series,
we have resorted to fluorination of the aminoisoquinoline P1
group to reduce its basicity. The novel 4-fluoroaminoisoquino-
line had a profound impact on pKa and thus permeability and
oral exposure. The resulting lead compound 13 achieved
clinical candidate level potency, selectivity, anticlotting activity,
and in vivo efficacy. It also achieved acceptable oral
bioavailability and half-life in dogs. The key limitation is poor
rodent metabolic stability and pharmacokinetics, preventing
further advancement.
EXPERIMENTAL SECTION
■
General Methods for Chemistry. All experiments were carried
out under an inert atmosphere and at room temperature unless
otherwise stated. Reactions were monitored using thin-layer
chromatography on 250 μm plates or using HPLC and LCMS with
UV detection at 220 or 254 nm. Purification was accomplished by
medium pressure liquid chromatography on a CombiFlash Com-
panion (Teledyne Isco) with RediSep normal phase silica gel or by
reverse phase preparative HPLC. All compounds for biological testing
were purified by preparative HPLC. Purity of all final compounds was
95% or higher, determined with two orthogonal HPLC conditions
(see Supporting Information for details). Reverse phase analytical and
preparative HPLC were carried out using Shimadzu HPLC system
running Discovery VP software. LCMS chromatograms were obtained
on a Shimadzu HPLC system running Discovery VP software, coupled
with a Waters ZQ mass spectrometer running MassLynx version 3.5
1
software. H NMR spectra were recorded on a Bruker spectrometer
(400 or 500 MHz) at ambient temperature.
(2R,15R)-2-[(1-Aminoisoquinolin-6-yl)amino]-4,15,17-trimethyl-
13-oxa-4,11-diazatricyclo[14.2.2.16,10]henicosa-1(18),6,8,10-
(21),16,19-hexaene-3,12-dione (1). Phosgene (0.12 mL, 0.19 mmol,
20% in toluene) was added dropwise to a solution of 20a (115 mg,
0.17 mmol) in MeCN (1.5 mL)/CH2Cl2 (1.5 mL) at 0 °C. The bath
was removed, and the mixture was stirred at room temperature for 30
min. Argon was bubbled through the solution to remove excess
phosgene, then the mixture was added to a solution of TEA (0.11 mL,
0.79 mmol) in CH2Cl2 (25 mL) at 40 °C over 5 h. The reaction was
stirred at room temperature overnight, quenched with H2O (1 mL)
and MeOH (5 mL), and concentrated. The crude product was purified
by flash chromatography (0−100% EtOAc/hexanes) to give a mixture
of diastereoisomers. The diastereoisomers were separated by a prep
(2R,15R)-2-[(1-Aminoisoquinolin-6-yl)amino]-N,N,4,15,17-pen-
tamethyl-3,12-dioxo-13-oxa-4,11-diazatricyclo[14.2.2.16,10]-
henicosa-1(18),6,8,10(21),16,19-hexaene-7-carboxamide (5). To a
solution of 24 (8 mg, 10.6 μmol) at 0 °C in DMF (0.3 mL) and
MeCN (0.3 mL) was added HATU (4.8 mg, 0.013 mmol) and N-
methylmorpholine (1.75 μL, 0.016 mmol). The reaction was stirred
G
DOI: 10.1021/acs.jmedchem.6b00469
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
for 5 min. Then 2 M dimethylamine in THF (6.4 μL, 0.013 mmol)
was added and the reaction was allowed to warm to room temperature
and stirred for 4 h. The reaction mixture was diluted with water and
extracted with EtOAc. The combined organic layer was washed with
water, brine, dried over MgSO4, and concentrated. The di-Boc
intermediate was treated with TFA (1.5 mL) for 30 min. The reaction
mixture was then concentrated and purified by prep HPLC to give 5
1.76 Hz, 1H), 7.47 (d, J = 8.35 Hz, 1H), 7.31 (d, J = 7.03 Hz, 1H),
7.17−7.24 (m, 2H), 6.92 (d, J = 7.03 Hz, 1H), 6.84 (d, J = 2.20 Hz,
1H), 6.76 (d, J = 7.91 Hz, 1H), 6.15−6.22 (m, 1H), 5.66−5.75 (m,
2H), 4.63 (t, J = 10.99 Hz, 1H), 4.21 (d, J = 17.14 Hz, 1H), 3.92−4.03
(m, 4H), 3.41−3.55 (m, 1H), 3.33 (br s, 3H), 2.33 (s, 3H), 1.24−1.38
(m, 6H). HPLC purity 99%.
(2R,15R)-2-[(1-Aminoisoquinolin-6-yl)amino]-7-(cyclopropane-
sulfonyl)-4,15,20-trimethyl-13-oxa-4,11-diazatricyclo[14.2.2.16,10]-
henicosa-1(18),6,8,10(21),16,19-hexaene-3,12-dione (10). In a
procedure similar to that described for 1, 20c (0.66 g, 0.83 mmol)
reacted with phosgene/TEA, diastereoisomers were chirally separated
(R,R-Whelk-01 column 21.1 mm × 250 mm), and the desired
enantiomer was deprotected with TFA to give 10 (115.8 mg, 38%
1
(2.9 mg, 39% yield). MS (ESI) m/z 581.2 [M + H]+. H NMR (400
MHz, MeOH-d4) δ ppm 8.06 (d, J = 9.29 Hz, 1H), 7.61−7.71 (m,
1H), 7.47 (d, J = 8.03 Hz, 2H), 7.33 (d, J = 7.03 Hz, 1H), 7.18−7.26
(m, 1H), 7.13 (d, J = 8.03 Hz, 1H), 6.93 (d, J = 7.03 Hz, 1H), 6.84 (d,
J = 2.26 Hz, 1H), 6.77 (dd, J = 8.03, 2.01 Hz, 1H), 6.14 (s, 1H), 5.75
(s, 1H), 5.27 (s, 1H), 4.60−4.73 (m, 1H), 4.01 (dd, J = 10.67, 4.39 Hz,
1H), 3.88 (d, J = 16.56 Hz, 1H), 3.44−3.58 (m, 1H), 3.27 (s, 3H),
3.11 (s, 3H), 2.94 (s, 3H), 2.35 (s, 3H), 1.33 (d, J = 7.03 Hz, 3H).
HPLC purity 98%.
1
yield) as white solid. MS (ESI) m/z 614.1 [M + H]+. H NMR (400
MHz, MeOH-d4) δ ppm 9.50 (s, 1H), 8.05 (d, J = 9.29 Hz, 1H), 7.73
(d, J = 8.53 Hz, 1H), 7.68 (dd, J = 7.91, 1.63 Hz, 1H), 7.48 (d, J = 8.03
Hz, 1H), 7.32 (d, J = 7.03 Hz, 1H), 7.20 (dd, J = 9.16, 2.38 Hz, 1H),
7.11 (s, 1H), 6.92 (d, J = 7.03 Hz, 1H), 6.80−6.86 (m, 2H), 6.42 (t, J
= 2.38 Hz, 1H), 5.78 (d, J = 17.57 Hz, 1H), 5.74 (s, 1H), 4.64 (t, J =
11.04 Hz, 1H), 4.30 (d, J = 17.57 Hz, 1H), 3.99 (dd, J = 10.79, 4.27
Hz, 1H), 3.46−3.55 (m, 1H), 3.40 (s, 3H), 2.81−2.92 (m, 1H), 2.31
(s, 3H), 1.34 (d, J = 7.03 Hz, 3H), 1.22−1.30 (m, 1H), 1.01−1.17 (m,
3H). HPLC purity 98%.
(2R,15R)-2-[(1-Amino-8-fluoroisoquinolin-6-yl)amino]-7-(cyclo-
propanesulfonyl)-4,15,17- trimethyl-13-oxa-4,11-diazatricyclo-
[14.2.2.16,10]henicosa-1(18),6,8,10(21),16,19-hexaene-3,12-dione
(11). In a procedure similar to that described for 1, 27a (0.2 g, 0.25
mmol) reacted with phosgene/TEA, diastereoisomers were chirally
separated (Chiral OD 10 um 4.6 mm × 250 mm; isocratic 35% 1:1
MeOH:EtOH/heptanes with 0.1% DEA), and the desired enantiomer
was deprotected with 4 N HCl in dioxane to give the 11 (7.7 mg, 10%
yield). MS (ESI) m/z 632.4 [M + H]+. 1H NMR (400 MHz, MeOH-
d4) δ ppm 7.71 (d, J = 8.34 Hz, 1H), 7.63 (dd, J = 7.83, 1.77 Hz, 1H),
7.54−7.41 (m, 2H), 7.10 (d, J = 1.52 Hz, 1H), 6.81 (dd, J = 8.46, 2.15
Hz, 1H), 6.78−6.64 (m, 2H), 6.51 (d, J = 2.27 Hz, 1H), 6.41 (d, J =
2.02 Hz, 1H), 5.75 (d, J = 17.43 Hz, 1H), 5.63 (s, 1H), 4.62 (t, J =
10.99 Hz, 1H), 4.28 (d, J = 17.68 Hz, 1H), 3.97 (dd, J = 10.86, 4.29
Hz, 1H), 3.55−3.44 (m, 1H), 3.40−3.36 (m, 4H), 2.29 (s, 3H), 1.32
(t, J = 7.33 Hz, 3H), 1.27−1.14 (m, 2H), 1.15−0.95 (m, 2H) 19F
NMR (376 MHz, MeOH-d4) δ ppm −115.25 (s, 1F). HPLC purity
98%.
(2R,15R)-2-[(1-Amino-7-fluoroisoquinolin-6-yl)amino]-7-(cyclo-
propanesulfonyl)-4,15,17-trimethyl-13-oxa-4,11-diazatricyclo-
[14.2.2.16,10]henicosa-1(18),6,8,10(21),16,19-hexaene-3,12-dione
(12). In a procedure similar to that described for 1, 27b (80 mg, 0.1
mmol) reacted with phosgene/TEA, diastereoisomers were chirally
separated (R,R-Whelk-01 column 21.1 mm × 250 mm), and the
desired enantiomer was deprotected with 4 N HCl in dioxane to give
the 12 (19 mg, 24% yield). MS (ESI) m/z 632.4 [M + H]+. 1H NMR
(400 MHz, MeCN-d3) δ ppm 7.92 (d, J = 12.64 Hz, 1H), 7.64−7.73
(m, 2H), 7.44 (d, J = 7.70 Hz, 1H), 7.29 (d, J = 7.15 Hz, 1H), 6.93−
6.99 (m, 2H), 6.90 (d, J = 7.15 Hz, 1H), 6.69−6.84 (m, 1H), 6.34 (s,
1H), 5.69−5.77 (m, 2H), 4.54−4.58 (m, 1H), 4.23 (d, J = 17.59 Hz,
1H), 3.91 (dd, J = 10.44, 4.40 Hz, 1H), 3.38−3.49 (m, 1H), 3.31 (s,
3H), 2.74−2.88 (m, 1H), 2.21 (s, 3H), 1.25 (d, J = 6.60 Hz, 3H),
0.97−1.13 (m, 4H). 19F NMR (376 MHz, MeCN-d3) δ ppm −131.68
(s, 1F). HPLC purity 99%.
(2R,15R)-2-[(1-Amino-4-fluoroisoquinolin-6-yl)amino]-7-(cyclo-
propanesulfonyl)-4,15,17-trimethyl-13-oxa-4,11-diazatricyclo-
[14.2.2.16,10]henicosa-1(18),6,8,10(21),16,19-hexaene-3,12-dione
(13). In a procedure similar to that described for 1, 27c (135 mg, 0.17
mmol) reacted with phosgene/TEA, diastereoisomers were chirally
separated (R,R-Whelk-01 column 21.1 mm × 250 mm), and the
desired enantiomer was deprotected with 4 N HCl in dioxane to give
the 13 (39 mg, 31% yield). MS (ESI) m/z 632.1 [M + H]+. 1H NMR
(400 MHz, MeCN-d3) δ ppm 7.74−7.81 (m, 2H), 7.71 (d, J = 8.35
Hz, 1H), 7.64−7.69 (m, 1H), 7.45 (d, J = 7.91 Hz, 1H), 7.21 (d, J =
4.83 Hz, 1H), 7.13 (dd, J = 9.23, 2.20 Hz, 1H), 6.98 (s, 1H), 6.88 (s,
1H), 6.81 (dd, J = 8.35, 1.76 Hz, 1H), 6.30 (d, J = 1.76 Hz, 1H), 5.70
(d, J = 17.14 Hz, 1H), 5.65 (d, J = 3.95 Hz, 1H), 4.56 (t, J = 10.99 Hz,
1H), 4.18 (d, J = 17.58 Hz, 1H), 3.91 (dd, J = 10.55, 4.39 Hz, 1H),
(2R,15R)-2-[(1-Aminoisoquinolin-6-yl)amino]-N,N-diethyl-
4,15,17-trimethyl-3,12-dioxo-13-oxa-4,11-diazatricyclo[14.2.2.16,10]-
henicosa-1(18),6,8,10(21),16,19-hexaene-7-carboxamide (6). In a
procedure similar to that described for 5, replacing dimethylamine
with diethylamine, 6 (3.4 mg, 29% yield) was obtained as a white solid.
1
MS (ESI) m/z 609.3 [M + H]+. H NMR (400 MHz, MeOH-d4) δ
ppm 7.99−8.11 (m, 1H), 7.65 (dd, J = 7.78, 1.76 Hz, 1H), 7.46 (d, J =
8.03 Hz, 1H), 7.31 (d, J = 7.03 Hz, 1H), 7.15−7.26 (m, 2H), 7.10 (d, J
= 8.03 Hz, 1H), 6.91 (d, J = 7.03 Hz, 1H), 6.83 (d, J = 2.26 Hz, 1H),
6.76 (dd, J = 8.03, 1.76 Hz, 1H), 6.10 (s, 1H), 5.73 (s, 1H), 5.25 (br s,
1H), 4.65 (t, J = 11.04 Hz, 1H), 3.99 (dd, J = 10.79, 4.27 Hz, 1H),
3.87 (br s, 2H), 3.59 (d, J = 1.00 Hz, 1H), 3.42−3.56 (m, 2H), 3.27 (s,
3H), 2.33 (s, 3H), 1.27−1.37 (m, 3H), 1.25 (t, J = 7.15 Hz, 3H), 1.10
(t, J = 7.03 Hz, 3H). HPLC purity 99%.
(2R,15R)-2-[(1-Aminoisoquinolin-6-yl)amino]-4,15,20-trimethyl-
7-(1-methyl-1H-pyrazol-5-yl)-13-oxa-4,11-diazatricyclo-
[14.2.2.16,10]henicosa-1(18),6,8,10(21),16,19-hexaene-3,12-dione
(7). In a procedure similar to that described for 1, 20e (200 mg, 0.26
mmol) reacted with phosgene/TEA, diastereoisomers were chirally
separated (R,R-Whelk-01 column 21.1 mm × 250 mm; isocratic 45%
MeOH/EtOH (1:1)/65% heptanes), and the desired enantiomer was
deprotected with TFA to give 7 (8.5 mg, 5% yield). MS (ESI) m/z
590.4 [M + H]+. 1H NMR (400 MHz, MeOH-d4) δ ppm 8.02 (d, J =
9.23 Hz, 1H), 7.55−7.79 (m, 2H), 7.39−7.56 (m, 2H), 7.29 (d, J =
7.03 Hz, 1H), 7.15−7.22 (m, 2H), 7.12 (d, J = 8.35 Hz, 1H), 6.88 (d, J
= 7.03 Hz, 1H), 6.80 (br s, 2H), 6.29 (d, J = 1.76 Hz, 1H), 6.17 (s,
1H), 5.70 (s, 1H), 4.98−5.14 (m, 2H), 4.16−4.25 (m, 2H), 3.98 (dd, J
= 10.55, 4.39 Hz, 1H), 3.67−3.70 (m, 3H), 3.67 (s, 3H), 3.49−3.54
(m, 3H), 2.33 (s, 3H). HPLC purity 96%.
Diethyl [(2R,15R)-2-[(1-Aminoisoquinolin-6-yl)amino]-4,15,17-tri-
methyl-3,12-dioxo-13-oxa-4,11-diazatricyclo[14.2.2.16,10]henicosa-
1(18),6,8,10(21),16,19-hexaen-7-yl]phosphonate (8). In a procedure
similar to that described for 1, 20b (130 mg, 0.16 mmol) reacted with
phosgene/TEA, diastereoisomers were chirally separated (Chiralcel
AD-H, 2.0 cm × 25 cm), and the desired enantiomer was deprotected
with 4.0 N HCl in dioxane to give 8 (24 mg, 22% yield) as a white
1
solid. MS (ESI) m/z 646.1 [M + H]+. H NMR (400 MHz, MeOH-
d4) δ ppm 9.39 (s, 1H), 8.04 (d, J = 9.23 Hz, 1H), 7.74 (dd, J = 14.06,
8.35 Hz, 1H), 7.67 (dd, J = 7.91, 1.32 Hz, 1H), 7.47 (d, J = 7.91 Hz,
1H), 7.31 (d, J = 7.03 Hz, 1H), 7.19 (dd, J = 9.23, 2.20 Hz, 1H), 7.16
(s, 1H), 6.92 (d, J = 7.03 Hz, 1H), 6.84 (m, 1H), 6.76−6.82 (m, 1H),
6.22−6.29 (m, 1H), 5.74 (s, 1H), 5.67 (d, J = 17.14 Hz, 1H), 4.63 (t, J
= 10.99 Hz, 1H), 4.06−4.14 (m, 4H), 3.96−4.02 (m, 1H), 3.43−3.55
(m, 1H), 3.35 (s, 3H), 2.30 (s, 3H), 1.29−1.38 (m, 9H). HPLC purity
95%.
[(2R,15R)-2-[(1-Aminoisoquinolin-6-yl)amino]-4,15,20-trimethyl-
3,12-dioxo-13-oxa-4,11-diazatricyclo[14.2.2.16,10]henicosa-1-
(18),6,8,10(21),16,19-hexaen-7-yl](ethoxy)phosphinic Acid (9). One
M LiOH (10 drops) was added to a solution of 8 (8 mg, 0.012 mmol)
in DMSO (4 drops) and THF (0.1 mL), and the mixture was stirred at
40 °C overnight. The reaction mixture was neutralized with 1.0 M HCl
(15 drops) and concentrated. The crude product was purified by prep
HPLC to yield 9 (4.0 mg, 51% yield) as a white solid. MS (ESI) m/z
618.5 [M + H]+. 1H NMR (400 MHz, MeOH-d4) δ ppm 8.05 (d, J =
9.23 Hz, 1H), 7.75 (dd, J = 13.84, 8.13 Hz, 1H), 7.67 (dd, J = 7.91,
H
DOI: 10.1021/acs.jmedchem.6b00469
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Journal of Medicinal Chemistry
Article
3.39 (ddd, J = 11.32, 7.14, 4.39 Hz, 1H), 3.27 (s, 3H), 2.63−2.72 (m,
1H), 2.17 (s, 3H), 1.25 (d, J = 7.03 Hz, 3H), 1.05−1.20 (m, 2H),
0.93−1.04 (m, 2H). 13C NMR (125.66 MHz, MeOH-d4) δ ppm
171.60, 154.81, 153.62, 152.98, 149.39, 145.12, 143.87, 139.51, 139.18,
134.39, 133.66, 133.08, 133.00, 129.10, 128.31, 128.10, 120.16, 119.80,
119.18, 113.35, 109.38, 99.33, 72.08, 58.15, 51.87, 37.81, 36.17, 33.56,
20.28, 15.72, 6.60, 6.18. 19F NMR (376 MHz, MeCN-d3) δ ppm
−154.37 (s, 1F). e.e. 99.8%. Anal. Calcd for C33H34N5O5SF·HCl: C,
55.60; H, 5.70; N, 9.64; S, 4.41; Cl, 5.30. Found: C, 56.79; H, 5.49; N,
9.50; S, 4.39; Cl, 5.20. HPLC purity 98%.
methylphenyl})methyl]amino}isoquinolin-1-yl)-N-[(tert-butoxy)-
carbonyl]carbamate (20e). In a procedure similar to that described
for 20d, replacing 21a with 21b, 20e (210 mg, 82% yield) was
1
obtained as a solid. MS (ESI) m/z 764.5 [M + H]+. H NMR was
complicated by a pair of diastereomers and rotamers.
tert-Butyl N-(6-{[(2R,15R)-7-Bromo-4,15,20-trimethyl-3,12-dioxo-
13-oxa-4,11-diazatricyclo[14.2.2.16,10]henicosa-1(18),6,8,10-
(21),16,19-hexaen-2-yl]amino}isoquinolin-1-yl)-N-[(tert-butoxy)-
carbonyl]carbamate (22). A solution of 15 (0.7 g, 1.9 mmol),
glyoxylic acid monohydrate 17 (0.18 g, 1.9 mmol), and 16 (0.38 g, 1.9
mmol) in DMF (3 mL) and MeCN (3 mL) was stirred at 80 °C for
1.5 h. The mixture was cooled to room temperature and diluted with
DMF (2 mL). To this mixture were added sequentially 4-bromo-3-
((methylamino)methyl)aniline (0.67 g, 2.3 mmol), BOP (0.94 g, 2.1
mmol), and TEA (1.6 mL, 11 mmol). The mixture was stirred at room
temperature for 30 min, then diluted with EtOAc, washed with water
and brine, and dried (Na2SO4). The solvent was removed under
reduced pressure. The residue was redissolved in CH2Cl2 with 3%
MeOH and purified by flash chromatography (1−10% MeOH/
CH2Cl2) to give the intermediate amide (1.2 g, 79% yield) as an
orange glass. MS (ESI) m/z 762.5 and 764.5 [M + H]+. 1H NMR was
complicated by a pair of diastereomers and rotamers.
tert-Butyl N-{6-[({[(3-Aminophenyl)methyl](methyl)carbamoyl}-
({4-[(2R)-1-hydroxypropan-2-yl]-3-methylphenyl})methyl)amino]-
isoquinolin-1-yl}-N-[(tert-butoxy)carbonyl]carbamate (20a). To a
mixture of 15 (120 mg, 0.33 mmol), 16 (64.8 mg, 0.33 mmol), and
glyoxylic acid monohydrate 17 (31 mg, 0.33 mmol) were added DMF
(1 mL) and MeCN (2 mL). The heterogeneous mixture was stirred at
80 °C for 3 h to give an orange solution of 18, which was cooled to
room temperature. To this mixture were added 19a (50.0 mg, 0.37
mmol), BOP (162 mg, 0.37 mmol), and TEA (0.23 mL, 1.7 mmol).
The mixture was stirred at room temperature for 1 h, diluted with
EtOAc, washed with water and brine, dried over Na2SO4, and
concentrated. The crude product was purified by flash chromatography
(1−10% MeOH/CH2Cl2) to afford 20a (117 mg, 51% yield) as off-
A solution of the above amide (1.17 g, 1.53 mmol) in MeCN (8
mL), CH2Cl2 (8 mL), and DMPU (0.6 mL) was cooled to 0 °C. To
this solution was added phosgene (20% in toluene, 0.84 mL, 1.69
mmol). The mixture was stirred at 0 °C for 15 min and then bubbled
with Ar for 10 min to remove excess phosgene. The resulting solution
was added dropwise over 3 h (via a syringe pump) into a solution of
TEA (2.1 mL, 15.4 mmol) in CH2Cl2 (400 mL) at 40 °C. The
solution was stirred for an additional 30 min. Reaction mixture was
concentrated, suspended in EtOAc (250 mL), washed with water and
brine, and dried (Na2SO4). EtOAc was removed under reduced
pressure, and the residue was purified by flash chromatography (50−
100% EtOAc/hexanes) to give product as a mixture of diastereomers.
The diastereoisomers were separated by chiral HPLC (Chiral OD 10
μm 4.6 mm × 250 mm; solvent A heptanes; solvent B 50% MeOH−
50% EtOH) to give 22 (0.2 g, 33% yield). MS (ESI) m/z 788.1 [M +
1
white solid. MS (ESI) m/z 684.5 [M + H]+. H NMR (500 MHz,
MeOH-d4) δ 8.03 (dd, J = 18.0, 5.9 Hz, 1H), 7.65−7.53 (m, 1H),
7.49−7.35 (m, 2H), 7.35−7.13 (m, 3H), 7.13−6.94 (m, 1H), 6.80−
6.44 (m, 4H), 5.64−5.48 (m, 1H), 4.65−4.45 (m, 2H), 3.69−3.64 (m,
1H), 3.58−3.53 (m, 1H), 3.22−3.15 (m, 1H), 3.05 (s, 3H), 2.39−2.34
1
(m, 3H), 1.30−1.25 (m, 18H), 1.24−1.20 (m, 3H). H NMR was
complicated by a pair of diastereomers and rotamers.
tert-Butyl N-(6-{[({[5-Amino-2-diethoxyphosphoryl)phenyl]-
methyl}(methyl)carbamoyl)({4-[(2R)-1-hydroxypropan-2-yl]-3-
methylphenyl})methyl]amino}isoquinolin-1-yl)-N-[(tert-butoxy)-
carbonyl]carbamate (20b). In a procedure similar to that described
for 20a, replacing 19a with 19b, 20b (135 mg, 71% yield) was
1
obtained as a yellow solid. MS (ESI) m/z 820.1 [M + H]+. H NMR
was complicated by a pair of diastereomers and rotamers.
1
tert-Butyl N-(6-{[({[5-Amino-2-(cyclopropanesulfonyl)phenyl]-
methyl}(methyl)carbamoyl)({4-[(2R)-1-hydroxypropan-2-yl]-3-
methylphenyl})methyl]amino}isoquinolin-1-yl)-N-[(tert-butoxy)-
carbonyl]carbamate (20c). In a procedure similar to that described
for 20a, replacing 19a with 19c, 20c (0.66 g, 77% yield) was obtained
as an orange glass, which was lyophilized to give a white powder. MS
H]+. H NMR (400 MHz, MeOH-d4) δ ppm 8.04 (d, J = 5.50 Hz,
1H), 7.68 (d, J = 6.05 Hz, 1H), 7.60 (d, J = 9.34 Hz, 1H), 7.51 (d, J =
6.05 Hz, 1H), 7.46 (d, J = 7.70 Hz, 1H), 7.40 (d, J = 8.24 Hz, 1H),
7.27 (dd, J = 8.79, 2.20 Hz, 1H), 7.22 (s, 1H), 6.87 (d, J = 2.20 Hz,
1H), 6.63 (dd, J = 8.52, 2.47 Hz, 1H), 5.99 (d, J = 2.20 Hz, 1H), 5.71
(s, 1H), 5.35 (d, J = 17.04 Hz, 1H), 4.65 (t, J = 11.27 Hz, 1H), 3.95
(dd, J = 10.99, 4.40 Hz, 1H), 3.65 (s, 1H), 3.44−3.53 (m, 1H), 3.34 (s,
3H), 2.31 (s, 3H), 1.31 (d, J = 7.15 Hz, 3H), 1.27 (s, 18H). HPLC
purity 95%.
1
(ESI) m/z 788.2 [M + H]+. H NMR was complicated by a pair of
diastereomers and rotamers.
tert-Butyl N-(6-{[({[5-Amino-2-(trifluoromethoxy)phenyl]methyl}-
(methyl)carbamoyl)({4-[(2R)-1-hydroxypropan-2-yl]-3-
methylphenyl})methyl]amino}isoquinolin-1-yl)-N-[(tert-butoxy)-
carbonyl]carbamate (20d). To a mixture of 15 (1.0 g, 2.8 mmol), 16
(0.54 g, 2.8 mmol), and glyoxylic acid monohydrate 17 (0.21 g, 2.8
mmol) were added DMF (6 mL) and MeCN (12 mL). The
heterogeneous mixture was stirred at 80 °C for 3 h to give an orange
solution of 18, which was cooled to room temperature. To this mixture
were added 21a (0.77 g, 2.7 mmol), BOP (1.49 g, 3.4 mmol), and
DIEA (1.2 mL, 6.8 mmol). The reaction was stirred at room
temperature overnight, diluted with EtOAc, washed with water and
brine, dried with Na2SO4, and concentrated. The crude product was
purified by flash chromatography (1−10% MeOH/CH2Cl2) to afford
the nitro intermediate (1.35 g, 63% yield) as a solid. MS (ESI) m/z
798.8 [M + H]+.
Methyl (2R,15R)-2-[(1-{Bis[(tert-butoxy)carbonyl]amino}-
isoquinolin-6-yl)amino]-4,15,17-trimethyl-3,12-dioxo-13-oxa-4,11-
diazatricyclo[14.2.2.16,10]henicosa-1(18),6,8,10(21),16,19-hexaene-
7-carboxylate (23). To a pressure flask charged with 22 (250 mg, 0.32
mmol), Pd(OAc)2 (35.6 mg, 0.16 mmol), TEA (0.13 mL, 0.95 mmol),
and 1,3-bis(diphenylphosphino)propane (dppp, 65.4 mg, 0.16 mmol)
were added DMSO (2 mL)/MeOH (1 mL). The resulting mixture
was charged with CO at 25 psi and then stirred at 80 °C under an
atmosphere of CO overnight. Reaction mixture was cooled to room
temperature, filtered, and concentrated. The resulting residue was
dissolved in EtOAc and washed with water. The organic layer was
washed with brine and dried (Na2SO4). The crude was purified flash
chromatography (1% MeOH/CH2Cl2) to give 23 (203 mg, 83%
yield). MS (ESI) m/z 768.4 [M + H]+. 1H NMR (400 MHz, MeOH-
d4) δ ppm 7.94 (d, J = 5.77 Hz, 1H), 7.81 (d, J = 8.28 Hz, 1H), 7.58
(d, J = 7.03 Hz, 1H), 7.49 (d, J = 9.03 Hz, 1H), 7.41 (d, J = 5.77 Hz,
1H), 7.36 (d, J = 7.78 Hz, 1H), 7.16 (dd, J = 9.16, 2.13 Hz, 1H), 7.08
(s, 1H), 6.78 (d, J = 1.76 Hz, 1H), 6.64 (dd, J = 8.28, 1.76 Hz, 1H),
6.14 (s, 1H), 5.54−5.66 (m, 2H), 4.52 (t, J = 10.92 Hz, 1H), 3.85 (dd,
J = 10.54, 4.27 Hz, 1H), 3.74 (s, 3H), 3.21 (d, J = 1.51 Hz, 2H), 3.20
(br s, 3H), 2.18 (s, 3H), 1.21 (d, J = 7.03 Hz, 3H), 1.15 (s, 18H).
(2R,15R)-2-[(1-{Bis[(tert-butoxy)carbonyl]amino}isoquinolin-6-
yl)amino]-4,15,17-trimethyl-3,12-dioxo-13-oxa-4,11-diazatricyclo-
[14.2.2.16,10]henicosa-1(18),6,8,10(21),16,19-hexaene-7-carboxylic
acid (24). To a solution of 23 (25 mg, 0.03 mmol) in THF (1 mL)
A mixture of the above nitro intermediate (1.34 g, 1.68 mmol),
4.0N HCl in dioxane (0.42 mL, 1.68 mmol), and 10% Pd/C (600 mg)
in MeOH (50 mL) was hydrogenated with a hydrogen balloon for 1.5
h. Pd/C was removed by filtration. The filtrate was concentrated and
redissolved in CH2Cl2 and washed with satd NaHCO3 and half-
saturated brine. The organic layer was dried over Na2SO4 and
concentrated to give 20d (1.2 g, 94% yield) as a yellow solid. MS
1
(ESI) m/z 768.4 [M + H]+. H NMR was complicated by a pair of
diastereomers and rotamers.
tert-Butyl-N-(6-{[({[5-amino-2-(1-methyl-1H-pyrazol-5-yl)phenyl]-
methyl}(methyl)carbamoyl)({4-[(2R)-1-hydroxypropan-2-yl]-3-
I
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Journal of Medicinal Chemistry
Article
was added 1.0 N LiOH (0.5 mL), and the reaction mixture was stirred
overnight. It was quenched with saturated NH4Cl, extracted with
EtOAC, washed with brine, and concentrated. The crude was purified
by prep HPLC to give 24 (14 mg, 57% yield). MS (ESI) m/z 754.3
[M + H]+.
6-Amino-1-(di-tert-butoxycarbonylamino)-8-fluoroisoquinoline
(25a). To a solution of 29a (21 mg, 0.06 mmol) in MeCN (2 mL) was
added BOC2O (58 mg, 0.27 mmol) followed by DMAP (5 mg, 0.04
mmol), then the suspension was stirred overnight at room
temperature. Solvent was removed under vacuum and the residue
purified by silica gel chromatography (0−30% MeOH/CH2Cl2) to
give the di-Boc intermediate (23 mg, 70% yield). MS (ESI) m/z 558.3
[M + H]+.
methylphenyl})methyl]amino}-7-fluoroisoquinolin-1-yl)-N-[(tert-
butoxy)carbonyl]carbamate (27b). In a procedure similar to that
described for 27a, replacing 25a with 25b, 27b (288 mg, 38% yield)
was obtained as a solid. MS (ESI) m/z 806.7 [M + H]+. 1H NMR was
complicated by a pair of diastereomers and rotamers. 19F NMR (376
MHz, MeCN-d3) δ ppm −130.36 (s, 1F).
tert-Butyl N-(6-{[({[5-Amino-2-(cyclopropanesulfonyl)phenyl]-
methyl}(methyl)carbamoyl)({4-[(2R)-1-hydroxypropan-2-yl]-3-
methylphenyl})methyl]amino}-4-fluoroisoquinolin-1-yl)-N-[(tert-
butoxy)carbonyl]carbamate (27c). In a procedure similar to that
described for 27a, replacing 25a with 25c, 27c (135 mg, 53% yield)
1
was obtained as a white solid. MS (ESI) m/z 806.6 [M + H]+. H
NMR was complicated by a pair of diastereomers and rotamers.
N6,N6-Dibenzyl-8-fluoroisoquinoline-1,6-diamine (29a). A mix-
ture of 28a (0.28 g, 0.79 mmol) and phosphorus oxychloride (3 mL)
was heated at 100 °C for 1 h. The reaction mixture was concentrated
under reduced pressure, then ice was added followed by 1.0 N sodium
hydroxide until the pH was basic. The resulting solid was collected by
filtration, washed with water, and dried in vacuo to give a chloride
intermediate (0.33 g) as a yellow solid. A portion of this solid (0.1 g)
was treated with a saturated solution of ammonia in ethylene glycol (4
mL) at 130 °C in an autoclave overnight. The mixture was
concentrated under reduced pressure, and the residue was purified
by silica gel chromatography (5% MeOH/CH2Cl2) to give 29a (55
To the above di-Boc intermediate (77 mg, 0.14 mmol) in ethanol
(20 mL) was added 20% palladium hydroxide on carbon (94 mg). The
reaction mixture was hydrogenated (55 psi) for 4 h. The mixture was
filtered and volatiles removed under reduced pressure to give 25a (47
1
mg, 90% yield) as a yellow solid. MS (ESI) m/z 378.3 [M + H]+. H
NMR (400 MHz, DMSO-d6) δ 8.05 (d, J = 6.0 Hz, 1H), 7.41 (dd, J =
6.0, 2.8 Hz, 1H), 6.79 (dd, J = 15.0, 2.0 Hz, 1H), 6.65 (d, J = 2.0 Hz,
1H), 6.26 (s, 2H), 1.30 (s, 18H).
6-Amino-1-(di-tert-butoxycarbonylamino)-7-fluoroisoquinoline
(25b). Using a procedure similar to that described for 25a, 29b (50
mg, 0.14 mmol) was Boc-protected and then hydrogenated to give 25b
(80 mg, 74% yield) as a white solid. MS (ESI) m/z 378.1 [M + H]+.
1H NMR (400 MHz, chloroform-d) δ ppm 8.25 (d, J = 5.77 Hz, 1H),
1
mg, 64% yield) as a brown solid. MS (ESI) m/z 358.4 [M + H]+. H
NMR (400 MHz, chloroform-d) δ 7.65 (d, J = 6.0 Hz, 1H), 7.38−7.32
(m, 4H), 7.31−7.20 (m, 6H), 6.68−6.60 (m, 2H), 6.55 (d, J = 2.7 Hz,
1H), 4.71 (s, 4H).
7.49 (d, J = 11.80 Hz, 1H), 7.37 (d, J = 5.77 Hz, 1H), 7.01 (d, J = 8.53
Hz, 1H), 4.34 (s, 2H), 1.35 (s, 18H).
N6,N6-Dibenzyl-7-fluoroisoquinoline-1,6-diamine (29b). Using a
procedure similar to that described for 29a, 28b (0.22 g, 0.61 mmol)
was chlorinated and then reacted with ammonia to give 29b (109 mg,
50% yield). MS (ESI) m/z 358.1 [M + H]+.
tert-Butyl N-(6-Amino-4-fluoroisoquinolin-1-yl)-N-[(tert-butoxy)-
carbonyl]carbamate (25c). To 34 (205 mg, 1.0 mmol) suspended in
MeCN (8.0 mL) were added TEA (0.55 mL, 4.0 mmol), DMAP (30
mg, 0.25 mmol), and Boc2O (1.0 M in THF, 3.0 mL, 3.0 mmol). The
cloudy mixture turned clear in 20 min. The reaction was continued at
room temperature for 6 h. It was then diluted with EtOAc, washed
with 0.5 N HCl, satd sodium carbonate, and brine, and dried over
Na2SO4. After evaporation of solvent, the residue was purified on silica
gel cartridge eluting with 0−30% EtOAc in hexanes to give the di-Boc
4-Fluoro-3-methoxy-6-nitro-3,4-dihydroisoquinolin-1(2H)-one
(31). To 30 (6.5 g, 34 mmol) in MeCN (180 mL) and MeOH (180
mL) was added Selectfluor (15.4 g, 44 mmol). The mixture was heated
at 82 °C for 1.5 h. Solvent was removed under vacuum. The crude was
suspended in EtOAc, stirred with 160 mL of 0.5 N HCl, and the
organic layer was collected. The aqueous layer was further extracted
with EtOAc, and the combined organic layers were washed with brine,
dried over Na2SO4, and concentrated to yield 31 (8.1 g, 99% yield) as
1
intermediate (266 mg, 66% yield) as a yellow solid. H NMR (400
MHz, MeOH-d4) δ ppm 9.06 (d, J = 2.20 Hz, 1H), 8.58 (dd, J = 9.23,
2.20 Hz, 1H), 8.51 (s, 1H), 8.27 (d, J = 9.23 Hz, 1H), 1.30 (s, 18H).
19F NMR (376 MHz, MeOH-d4) δ ppm −136.64 (s, 1F).
1
a brown solid. MS (ESI) m/z 241.4 [M + H]+. H NMR (400 MHz,
chloroform-d) δ ppm 8.56 (br, 1H), 8.11−8.38 (m, 3H), 5.83−5.99
(dd, J = 47.8, 3.85 Hz, 1H) and 5.38−5.54 (dd, J = 48.37, 2.2 Hz,1H),
4.89−4.97 (m, 2H) 3.39 and 3.38 (s, 3H). 19F NMR (376 MHz,
chloroform-d) δ −168.64 (d, J = 46.24 Hz) and −201.6 (d, J = 52.00
To the above di-Boc intermediate (266 mg, 0.65 mmol) in MeOH
(8 mL) was added 10% Pd/C (150 mg) and 1.0 N HCl (0.075 mL,
0.075 mmol). The mixture was placed under a hydrogen balloon and
stirred for 30 min. Pd/C was removed by filtration. and the filtrate was
concentrated to give 25c (245 mg, 99% yield) as a yellow solid. MS
1
Hz). H and 19F NMR indicated a mixture of two diastereoisomers.
4-Fluoro-6-nitroisoquinolin-1(2H)-one (32). To a solution of 31
(8.1 g, 33.7 mmol) in MeCN (100 mL) was added 4.0 N HCl in
dioxane (25.3 mL, 101 mmol). The mixture was stirred at 65 °C for
1.5 h. Solvent was removed under reduced pressure to yield 32 (8.1 g,
98% yield) as a yellow solid. MS (ESI) m/z 209.2 [M + H]+. 1H NMR
(400 MHz, DMSO-d6) δ ppm 8.38−8.46 (m, 2H), 8.31 (dd, J = 8.79,
2.20 Hz, 1H), 7.61 (d, J = 2.20 Hz, 1H). 19F NMR (376 MHz, DMSO-
d6) δ ppm −160.17 (s, 1F).
1
(ESI) m/z 378.3 [M + H]+. H NMR (400 MHz, MeCN-d3) δ ppm
7.96 (d, J = 3.08 Hz, 1H), 7.65 (dd, J = 9.23, 2.20 Hz, 1H), 7.13 (dd, J
= 9.23, 2.20 Hz, 1H), 6.95 (d, J = 2.20 Hz, 1H), 1.27 (s, 18H). 19F
NMR (376 MHz, MeCN-d3) δ ppm −142.33 (s, 1F).
tert-Butyl N-(6-{[({[5-Amino-2-(cyclopropanesulfonyl)phenyl]-
methyl}(methyl)carbamoyl)({4-[(2R)-1-hydroxypropan-2-yl]-3-
methylphenyl})methyl]amino}-8-fluoroisoquinolin-1- yl)-N-[(tert-
butoxy)carbonyl]carbamate (27a). 25a (400 mg, 1.06 mmol), 16
(247 mg, 1.27 mmol), and glyoxylic acid monohydrate 17 (98 mg,
1.06 mmol) were dissolved in DMF (2 mL) and MeCN (4 mL). The
reaction mixture was heated at 100 °C in the microwave for 10 min to
generate a solution of 26a, then allowed to cool to room temperature.
To this solution were added sequentially 19c (398 mg, 1.27 mmol),
BOP (563 mg, 1.27 mmol), and TEA (1.48 mL, 10.6 mmol). The
mixture was stirred at room temperature for 18 h. The reaction
mixture was quenched with water (0.5 mL), diluted with EtOAc (150
mL), washed with water and brine, and dried (Na2SO4). The solvent
was removed under reduced pressure. The crude product was purified
by flash chromatography (0−100% EtOAc/hexanes) to give 27a (460
mg, 54% yield) as an orange glass, which was lyophilized to give a
powder. MS (ESI) m/z 806.7 [M + H]+. 1H NMR was complicated by
a pair of diastereomers and rotamers.
1-Chloro-4-fluoro-6-nitroisoquinoline (33). To 32 (8.8 g, 36.0
mmol) was added phosphoryl trichloride (59.3 mL, 648 mmol). The
suspension was heated at 115 °C for 1.0 h. Solvent was removed under
high vacuum. The residue was diluted with EtOAc, washed with satd
NaHCO3 and brine, and dried over Na2SO4. After evaporation of
solvent, the residue was purified on silica gel cartridge which was
eluted with EtOAc in hexanes using 15 min gradient from 2% to 25%
to give 33 (8.5 g, 100% yield) as a slightly yellow solid. MS (ESI) m/z
1
227.1 [M + H]+. H NMR (400 MHz, chloroform-d) δ ppm 9.01 (s,
1H), 8.46−8.56 (m, 2H), 8.34 (s, 1H). 19F NMR (376 MHz,
chloroform-d) δ ppm −136.54 (s, 1F).
4-Fluoro-6-nitroisoquinolin-1-amine (34). A mixture of 33 (0.57 g,
2.5 mmol), BINAP (0.16 g, 0.25 mmol), and Pd(OAc)2 (0.028 g, 0.13
mmol) in toluene (12 mL) was degassed with Ar for 10 min. To this
mixture was added benzophenone imine (0.54 mL, 3.2 mmol) and
Cs2CO3 (312 mg, 3.2 mmol). The mixture was heated at 90 °C
tert-Butyl N-(6-{[({[5-Amino-2-(cyclopropanesulfonyl)phenyl]-
methyl}(methyl)carbamoyl)({4-[(2R)-1-hydroxypropan-2-yl]-3-
J
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Journal of Medicinal Chemistry
Article
overnight. After cooling to room temperature, it was diluted with
EtOAc/water/brine and filtered through a pad of wet Celite. The
organic layer was collected and washed with brine, dried over Na2SO4,
and concentrated to give the crude imine. The crude imine was
dissolved in THF (15 mL) and treated with 4.0 N HCl (9.6 mL, 38
mmol) for 30 min. The mixture was diluted with EtOAc, the aqueous
was collected, and the organic was further extracted with 4.0 N HCl (2
× 10 mL). The aqueous layers were combined and treated at 0 °C
with 5.0 N NaOH to adjust the pH to 12−13. The precipitate formed
was then collected by filtration and dried under a vacuum oven. It was
then redissolved in MeOH/CH2Cl2 and evaporated to dryness to give
34 (0.41 g, 79% yield) as a brown solid. (ESI) m/z 208.1 [M + H]+.
1H NMR (400 MHz, DMSO-d6) δ ppm 8.59 (d, J = 2.20 Hz, 1H),
8.51 (dd, J = 9.34, 2.20 Hz, 1H), 8.29 (dd, J = 9.34, 2.20 Hz, 1H), 7.99
(d, J = 2.20 Hz, 1H), 7.16 (s, 2H). 19F NMR (376 MHz, MeOH-d4) δ
−156.53 (s, 1F).
Enzyme Affinity Assays. Factors IXa, Xa, XIa, and activated
protein C (aPC) were purchased from Haematologic Technologies.
Factor XIIa, plasmin, and recombinant single chain tissue-type
plasminogen activator (tPA) were purchased from American
Diagnostica. Plasma kallikrein and α-thrombin were purchased from
Enzyme Research Laboratories. Urokinase was purchased from Abbott
Laboratories. Tissue kallikrein-1 (HK-1) was purchased from Dr. Julie
Chao, Medical University of South Carolina. Trypsin and chymo-
trypsin were purchased from Sigma-Aldrich. Recombinant factor VIIa
was purchased from Novo Nordisk. Recombinant soluble tissue factor
residues 1−219 were produced at Bristol-Myers Squibb.
was determined by fitting data from independent measurements at
several substrate concentrations to the Michaelis−Menten equation: v
= (Vmax*[S])/(Km + [S]) where v is the observed velocity of the
reaction, Vmax is the maximal velocity, [S] is the concentration of
substrate, and Km is the Michaelis constant for the substrate.
Values of IC50 were determined by allowing the protease to react
with the substrate in the presence of the inhibitor. Reactions were
allowed to go for periods of 10−120 min (depending on the protease)
and the velocities (rate of absorbance or fluorescence change versus
time) were measured.
The following relationships were used to calculate IC50 values: vs/vo
= A + ((B − A)/(1 + (IC50/I)n)), where vo is the velocity of the
control in the absence of inhibitor, vs is the velocity in the presence of
inhibitor, I is the concentration of inhibitor, A is the minimum activity
remaining (usually locked at zero), B is the maximum activity
remaining (usually locked at 1.0), n is the Hill coefficient, a measure of
the number and cooperativity of potential inhibitor binding sites, and
IC50 is the concentration of inhibitor that produces 50% inhibition.
When negligible enzyme inhibition was observed at the highest
inhibitor concentration tested, the value assigned as a lower limit for
IC50 is the value that would be obtained with either 25% or 50%
inhibition at the highest inhibitor concentration. In all other cases, IC50
values represent the average of duplicate determinations obtained over
8−11 concentrations. The intraassay and interassay variabilities are 5%
and 20%, respectively. Competitive inhibition was assumed for all
proteases. IC50 values were converted to Ki values by the relationship:
Ki = IC50/(1 + [S]/Km)
(1)
Factor XIa, factor XIIa, chymotrypsin, tPA, plasmin, and urokinase
assays were conducted in 50 mM HEPES, pH 7.4, 145 mM sodium
chloride, 5 mM potassium chloride, and 0.1% PEG 8000. Factor Xa,
thrombin, trypsin, plasma kallikrein, HK-1, and aPC assays were
conducted in 100 mM sodium phosphate, pH 7.4, 200 mM sodium
chloride, and 0.5% PEG 8000. Factor VIIa assays were conducted in 50
mM HEPES, pH 7.4, 150 mM sodium chloride, 5 mM calcium
chloride, and 0.1% PEG 8000. Factor IXa assays were conducted in 50
mM TRIS, pH 7.4, 100 mM sodium chloride, 5 mM calcium chloride,
0.5% PEG 8000, and 2% DMSO. The peptide substrates were: pyro-
Glu-Pro-Arg-pNA(para-nitroaniline) (Diapharma) for factor XIa,
thrombin, and aPC; N-benzoyl-Ile-Glu-(OH,OMe)-Gly-Arg-pNA
(Diapharma) for factor Xa and trypsin; methylsulfonyl-D-cyclo-
hexylglycyl-Gly-Arg-AMC(7-amino-4-methylcoumarin) (Pentapharm)
for factor IXa; H-(D)-Ile-Pro-Arg-pNA (Diapharma) for factor VIIa;
H-(D)-CHT-Gly-Arg-pNA (American Diagnostica) for factor XIIa; H-
(D)-Pro-Phe-Arg-pNA (Diapharma) for plasma kallikrein; MeO-Suc-
Arg-Pro-Tyr-pNA (Diapharma) for chymotrypsin; H-(D)-Val-Leu-Lys-
pNA (Diapharma) for plasmin; Methylsulfonyl-D-cyclohexylalanyl-
Gly-Arg-pNA (American Diagnostica) for tPA; pyro-Glu-Gly-Arg-pNA
(Diapharma) for urokinase; H-D-Val-Leu-Arg-AFC(7-amino-4-trifluor-
omethylcoumarin) (Calbiochem) for tissue kallikrein-1.
FVIIa-Xase Ki (37 °C) Determination. S2765 (0.5 mM), PCPS
(phosphatidylcholine phosphatidylserine, 25 μM), calcium chloride (5
mM), full-length human TF (3 nM), human FVIIa (5 pM), and FVIIa
inhibitor were incubated for 15 min at 37 °C. Reactions were initiated
by the addition of human FX (range of concentrations). Preliminary
experiments revealed that the plasma purified FX contains a residual
amount of human FVIIa which could not be removed by affinity
chromatography. The residual FVIIa is sufficient when combined with
PCPS, calcium, and TF to catalyze the conversion of FX to Xa and was
increased by the addition of 5 pM human FVIIa. FXa in turn
hydrolyses S2765, which was monitored for 60 min at 405 nm. FXase
activity was derived from the parabolic change in absorbance over time
according to eq 2:
absorbance = 1/2at2 + bt + c
(2)
where a is proportional to the rate of FX activation ≡ product ≡ vs, b is
proportional to the hydrolysis of S2765 in the absence of FXa, and c is
the absorbance at t = 0. Steady-state reaction velocity data (vs) was
globally fit to eq 3 for noncompetitive inhibition modified for
contaminating enzyme (FVIIa) in the substrate (FX) as
All assays were conducted at room temperature except where noted
in 96-well microtiter plate spectrophotometers or spectrofluorimeters
(Molecular Devices) with simultaneous measurement of enzyme
activities in control and inhibitor containing solutions. Compounds
were dissolved and diluted in DMSO and analyzed at a final
concentration of 1% DMSO except where noted. Assays were initiated
by adding enzyme to buffered solutions containing substrate in the
presence or absence of inhibitor. Hydrolysis of the substrate resulted
in the release of pNA (para-nitroaniline), which was monitored
spectrophotometrically by measuring the increase in absorbance at 405
nm, or the release of AMC (7-amino-4-methylcoumarin), which was
monitored spectrofluorometrically by measuring the increase in
emission at 460 nm with excitation at 380 nm, or the release of
AFC (7-amino-4-trifluoromethylcoumarin), which was monitored
spectrofluorometrically by measuring the increase in emission at 505
nm with excitation at 400 nm. The rate of absorbance or fluorescence
change is proportional to enzyme activity. A decrease in the rate of
absorbance or fluorescence change in the presence of inhibitor is
indicative of enzyme inhibition. Assays were conducted under
conditions of excess substrate and inhibitor over enzyme. The
Michaelis constant, Km, for substrate hydrolysis by each protease
v = (kcat(E + f[S])/((Km + [S])(1 + [I]/Ki))
(3)
s
where kcat is the enzyme turnover rate, E is the concentration of
enzyme added, f is the molar fraction of enzyme contained in the
substrate, and the rest as defined above.
Anticlotting Assays. Anticlotting assays were performed in a
temperature-controlled automated coagulation device (Sysmex CA-
6000 or CA-1500, Dade-Behring). Blood was obtained from healthy
volunteers by venipuncture and anticoagulated with one-tenth volume
0.11 M buffered sodium citrate (Vacutainer, Becton Dickinson).
Plasma was obtained after centrifugation at 2000g for 10 min and kept
on ice prior to use. An initial stock solution of the inhibitor at 10 mM
was prepared in DMSO. Subsequent dilutions were done in plasma.
Clotting time was determined on control plasma and plasma
containing up to seven different concentrations of inhibitor. FVIIa
deficient PT (FVII def PT) was performed by mixing human FVII
immunodepleted plasma with normal pooled plasma from different
species to produce a clotting time of about 40 seconds (s). Plasma (50
μL) was warmed to 37 °C for 3 min before adding thromboplastin
(100 μL, Thromboplastin C Plus, Dade-Behring, Illinois) to initiate
coagulation. Determinations were performed in duplicate and
K
DOI: 10.1021/acs.jmedchem.6b00469
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
expressed as a mean ratio of treated vs baseline control. The
concentrations required to prolong clotting time by 2-fold (EC2x) were
calculated by linear interpolation (Microsoft Excel, Redmond, WA,
USA) and are expressed as total plasma concentrations, not final assay
concentrations after addition of clotting assay reagents.
measurement. We thank colleagues at the BMS-Biocon
Research Center for synthesis of several intermediates.
ABBREVIATIONS USED
■
In Vivo Electrically Induced Carotid Artery Thrombosis
(ECAT) Model. The in vivo studies in rabbit ECAT model and the
calculation of % thrombus reduction were performed according to the
published protocols.11 Male New Zealand White rabbits were
anesthetized with ketamine (50 mg/kg + 50 mg/kg/h IM) and
xylazine (10 mg/kg + 10 mg/kg/h IM). These anesthetics were
supplemented as needed. An electromagnetic flow probe was placed
on a segment of an isolated carotid artery to monitor blood flow. Test
agents or vehicle was administered by intravenous (IV) infusion prior
to or after the initiation of thrombosis. Drug treatment prior to
initiation of thrombosis was used to model the ability of test agents to
prevent and reduce the risk of thrombus formation, whereas dosing
after initiation was used to model the ability to treat existing
thrombotic disease. Thrombus formation was induced by electrical
stimulation of the carotid artery for 3 min at 4 mA using an external
stainless steel bipolar electrode. Carotid blood flow was measured
continuously over a 90 min period to monitor thrombus-induced
occlusion. Total carotid blood flow over 90 min is calculated by the
trapezoidal rule. Average carotid flow over 90 min is then determined
by converting total carotid blood flow over 90 min to percent of total
control carotid blood flow, which would result if control blood flow
had been maintained continuously for 90 min. The EC50 (dose that
increased average carotid blood flow over 90 min to 50% of the
control) of compounds are estimated by a nonlinear least-squares
regression program using the Hill sigmoid Emax equation (DeltaGraph;
SPSS Inc., Chicago, IL).
TF-FVIIa, tissue factor−factor VIIa; FIXa, factor IXa; FXa,
factor Xa; FXIa, factor XIa; FXIIa, factor XIIa; aPC, active
protein C; HK-1, human tissue kallikrein 1; tPA, tissue
plasminogen activator; ECAT, electrically induced carotid
arterial thrombosis; dppp, 1,3-bis(diphenylphosphino)propane;
HATU, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo-
[4,5-b]pyridinium 3-oxid hexafluorophosphate
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%thrombus reduction
= (1 − thrombus weight after treatment
/control thrombus weight)× 100%
(4)
EC50 was determined using the four-parameter logistic equation y =
A + ((B − A)/(1 + ((C/x)D))), where A = minimum y value, B =
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.6b00469.
■
S
Descriptions of biological assays, syntheses and charac-
terization data for 19b,c and 21a,b, key compound
characterization of 13. (PDF)
Molecular formula strings (CSV)
AUTHOR INFORMATION
Corresponding Author
*Phone: 609-466-5104. Fax: 609-818-3460. E-mail: xiaojun.
zhang@bms.com.
■
Notes
The authors declare no competing financial interest.
§Yan Zou was deceased on March 21, 2014
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ACKNOWLEDGMENTS
■
We thank Jeff Bozarth, Randi Brown, Sara Peterson, and
Mojgan Abousleiman for obtaining the enzyme inhibition data,
and Frank Barbera and Yiming Wu for obtaining the clotting
assay data. We also thank Atsu Apedo, Gottfried Wenke, and
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J. Med. Chem. XXXX, XXX, XXX−XXX

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